Common pregnancy complications, such as intrauterine growth restriction (IUGR) and miscarriage, are strongly associated with abnormal development of the placenta. Defective placentation may result from exogenous environmental or maternal factors or from inborn genetic anomalies. However, our understanding of the cellular basis of IUGR or miscarriage is blocked by our ignorance about the process of normal placental development. We are cognizant that normal human placental development is dependent on differentiation of the villous cytotrophoblast culminating in intercellular fusion into the expanding syncytiotrophoblast on the placental surface. Syncytialization involves several processes, particularly cessation of cellular proliferation, redistribution of plasma membrane phospholipids to form a phosphatidylserine (PS)-rich exofacial surface, expression and insertion of fusion proteins into the plasma membrane, and rearrangement of actin cytoskeletal elements. Our preliminary data suggest that several apoptosis-related proteins, particularly caspases 3, 8, and 14, may be critical participants in trophoblast differentiation. The aim of this application is to investigate the role of these caspases in villous cytotrophoblast differentiation. We will use both primary villous cytotrophoblast cultures and a BeWo choriocarcinoma model. Using peptide inhibitors and gene silencing, we will determine the role of caspases 3, 8, and 14 in individual components of the syncytialization process (PS efflux, G1 arrest, cytoskeletal rearrangement, fusion protein expression, hCG production). Additional studies will evaluate the role of signal transduction through NF[unreadable]B. The ultimate goal of this investigation is to identify the critical components controlling the mechanism of villous cytotrophoblast differentiation. Unexplained IUGR and miscarriage are common complications that lead to death of a child or to a severely compromised newborn rekeletal elements. Our preliminary data suggest that several apoptosis-related proteins, particularly caspases 3, 8, and 14, may be critical participants in trophoblast differentiation. The aim of this application is to investigate the role of these caspases in villous cytotrction of hCG to maintain production of progesterone by the corpus luteum early in pregnancy and transport of gasses, nutrients and maternal protective antibodies across the placenta, and many other functions related to a healthy pregnancy. Expansion and maintenance of the syncytiotrophoblast, and therefore continued fetal well-being, is dependent on continuous intercellular fusion throughout most of pregnancy. Abnormalities in placental development and function are components of most complications of pregnancy, including miscarriage, preeclampsia, intrauterine growth restriction (IUGR), and preterm labor. There is a marked imbalance between the importance of successful syncytiotrophoblast formation and our knowledge of the process. Those working in the area would agree that syncytialization is dependent on villous cytotrophoblast differentiation, particularly cessation of cellular proliferation (i.e., a state of G1 arrest), redistribution of plasma membrane phospholipids to form an aminophospholipid (particularly phosphatidylserine [PS])-rich exofacial surface, expression and insertion of fusion proteins into the plasma membrane, and membrane joining mediated by breakdown and reconstruction of actin cytoskeletal elements. Differentiation of villous cytotrophoblast is also associated with alterations in the level and activity of several apoptosis-related proteins. Despite major controversy concerning the precise timing and nature of the changes, syncytialization is concurrent with diminished levels of pro-apoptotic proteins (e.g., caspases 3 and 8), increases in some anti-apoptotic proteins (e.g., Bcl-2), and apparent increased resistance to apoptosis. Because some components of cytotrophoblast fusion (e.g., PS efflux, G1 arrest, cytoskeletal changes) are common to cells undergoing apoptosis, there is considerable interest in whether apoptosis contributes to villous cytotrophoblast differentiation. Some have proposed that villous cytotrophoblast fusion results from aborted activation of apoptotic pathways, particularly through caspase 8-mediated PS efflux. As will be discussed in the Background and Significance portion of this proposal, that hypothesis is highly controversial with considerable data supporting both apoptotic- and non-apoptotic-related mechanisms of PS efflux. This controversy has resulted in an impasse to further understanding the process of normal placental development and how it may be adversely affected in complications of pregnancy. The purpose of this RO1 application is to investigate conflicting theories on the relationship between intertrophoblast fusion and alterations in apoptotic proteins. We propose the following working hypothesis that may explain apparently contradictory observations. Successful syncytiotrophoblast formation requires several cellular changes (e.g., entrance into a non-proliferative state, efflux of PS, and cytoskeletal rearrangement) that are regulated independently;some through atypical activation of the apoptosis cascade. We propose to test this hypothesis from two directions. AIM 1: Is syncytialization dependent on early apoptotic processes? Our 2 year Aim will expand our preliminary studies and focus on the role of three caspases (caspases 3, 8, and 14) in villous cytotrophoblast differentiation. We will study individual components of the differentiation/syncytialization process (PS efflux, G1 arrest, cytoskeletal rearrangement, fusion protein expression, hCG production) using in vitro models of villous cytotrophoblast differentiation and fusion (primary villous cytotrophoblast cultures, BeWo choriocarcinoma). In our most recent preliminary studies, we observed that decreased transcription and translation of caspase 3 and caspase 8 and increased expression of caspase 14 were concurrent with differentiation/syncytialization. Each of these procaspases has been linked by non-apoptotic mechanisms to control of cellular differentiation and proliferation in other cell types. Silencing of procaspase 8 in our preliminary studies resulted in significantly delayed differentiation and syncytialization. Experiments in this Aim will more thoroughly describe correlations between differentiation/syncytialization and alterations in protein and mRNA of these caspases as well as potential collaborative proteins, such as c-FLIPL and FADD. We will investigate those correlations by specifically inhibiting these proteins using peptide inhibitors and gene silencing. Additional studies will evaluate the role of signal transduction through NF[unreadable]B.